IgE ANTIBODIES FOR THE TREATMENT OF CANCER

ABSTRACT

The present invention provides monoclonal IgE antibodies comprising Fc epsilon (ε) constant regions and variable regions comprising at least one antigen binding region specific for binding a single epitope of a circulating, tumor-associated antigen (TAA) that is not a cell surface antigen wherein the epitope of the targeted antigen is not highly repetitive or is a non-repetitive epitope. The IgE antibodies of the invention are useful in the treatment of cancer associated with the tumor antigen. In one embodiment the TAA is prostate-specific antigen (PSA).

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2009/040085, which designated the United States and was filed onApr. 9, 2009, published in English, which claims the benefit of U.S.Provisional Application Nos. 61/043,682, filed on Apr. 9, 2008,61/044,576, filed on Apr. 14, 2008 and 61/159,069, filed on Mar. 10,2009. The entire teachings of the above applications are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

The newly arising field of AllergoOncology is based upon observationsand studies showing that those individuals with raised levels of IgE(e.g. individuals who suffer from allergies) are much less likely tosuffer from certain types of cancer. Researchers in this field areexploring the therapeutic potential of the IgE antibody class in theprevention and treatment of certain cancers, under the premise thatredirected immune pathways developed as adaptive responses tomicrobial/parasitic infection might be directed against malignancy.

IgE antibodies mediate allergic and asthmatic reactions, characterizedby immediate hypersensitivity, followed by an inflammatory delayed typeresponse requiring the recruitment of effector cells. The uniqueness ofthe allergic reaction is due to the presence of mast cells andLangerhans/dendritic cells in the tissue that are sensitized by the IgEbound to the high-affinity FcεRI (Kinet, J P, Annu. Rev. Immunol., 17:931-72: 931-972 (1999); and Ravetch J V, and Kinet J P, Annu. Rev.Immunol., 9: 457-492 (1991)). The activated Langerhans/dendritic cellsmigrate to local lymph nodes and stimulate cognate T cells, whichmigrate to the tissue, participate in the inflammatory response andstimulate antibody synthesis. IgE bound to mast cells and basophils cancause degranulation of the cells, but it requires cross-linking by theantigen the IgE recognizes. Following the acute phase of recruitment,eosinophils are recruited in the late-phase reaction. Activatedeosinophils are strong mediators of antibody-dependent cell-mediatedcytotoxicity (ADCC) via toxic granule proteins and cause tissue damagevia pro-inflammatory cytokines and vasoactive lipid mediators(leukotrienes, prostaglandin D2, platelet-activating factor). Theprocessing of the IgE containing immune complexes by Langerhans cellsand dendritic cells is a critical step for the induction of thelate-phase reaction. Activated T helper cells generate IL-4 and IL-5,which in turn recruits and activates eosinophils causing ADCC andantibody-dependent cell-mediated phagocytosis (ADCP) (Kinet, J P, Annu.Rev. Immunol., 17:931-72.:931-972 (1999); Maurer, D., et al., J.Immunol., 161: 2731-2739 (1998) and Maurer D., et al., J. Immunol., 154:6285-6290 (1995)).

While B cells can recognize antigen in its native conformation, T cellsgenerally recognize antigen that has been “processed” by antigenpresenting cells and then presented on the surface of the cell by majorhistocompatibility complex (MHC) molecules (Peakman, M. and Vergani, D.,New York: Churchhill Livingston; (1997)). MHC molecules are receptorsfor peptide antigens. There are two classes of MHC molecules, termed MHCclass I and MHC class II. Although united in their function of peptideantigen presentation and contact points for T cells, the differences inthe structure and intracellular trafficking of the two types arecritical because among other things, they elicit very different immuneresponses. A major obstacle in the creation of effective tumor immunityis that typically, there is poor presentation of tumor antigen on MHCclass I and class II molecules together (cross-presentation). Dendriticcells are bone marrow-derived leukocytes that are more potent initiatorsof T cell-dependent immune responses than any other antigen presentingcells that have been tested (Peakman, M. and Vergani, D., New York:Churchill Livingston; (1997)). Unlike other antigen presenting cells,dendritic cells can acquire antigens from their environment and processthem for cross-presentation, allowing activation of both CD8⁺ and CD4⁺ Tcells. However, this process requires high antigen concentrations.Simultaneous presentation on MHC II provides for T helper cellactivation. Depending on the stimuli, either production of cytokinesIL-12 and IFN-γ by T helper (Th) cell 1 type and cytotoxic T lymphocytes(CTL) induction occurs (collectively referred to herein as the “Th1/Tc1immune response; or IL-4, IL-5 and IL-10 is produced by Th2 cells for Bcell help (referred to herein as “Th2 immune response”). An importantfactor in immune induction is the activation or maturation of theantigen presenting cells, which induces the expression of co-stimulatorymolecules that are necessary to engage the T cell.

It is now believed that the engagement of the toll-like receptor (TLR)family (Okamoto, M. and Sato, M., J. Med. Invest., 50: 9-24 (2003)) aswell as other receptors including Fc receptors (Hamano, Y., et al., J.Immunol., 164: 6113-6119 (2000) and Regnault, A., et al., The Journal ofExperimental Medicine, 189: 371-380 (1999)) mediates activation andmaturation of macrophages and dendritic cells, which is crucial foractivating the innate immune system. Fc receptors have also been shownto facilitate antigen uptake and presentation. Researchers have shownthat immune complex pulsed DC induce stronger CD4⁺ and CD8⁺ T cellresponses as compared to DC pulsed with PSA alone (Berlyn, K A, et al.,Clin. Immunol., 101: 276-283 (2001). Similarly, NY-ESO-1 as well asovalbumin or pyruvate dehydrogenase are all presented to T cells muchmore efficiently when captured as an immune complexes rather than asfree-antigen (Regnault A., et al., The Journal of Experimental Medicine189:371-380 (1999); Nagata Y., et al., Proc. Natl. Acad. Sci. U.S.A.,99: 10629-10634 (2002); Kita, H., et al., J. Exp. Med., 195:113-123(2002) and Schuurhuis, D H, et al., J. Immunol., 168: 2240-2246 (2002)).The results suggest that effective cancer vaccines may be generated byadministering antibodies that target circulating antigen and form immunecomplexes that target DC in vivo. The role of IgE as a component of animmune complex in altering antigen presentation is less understood andis believed to contribute to the aggravation and perpetuation of theatopic response to allergen as demonstrated in the IgE mediatedinfluence enhancing Th2 (IL-4, IL-10) T cells responses to allergen.(Maurer et al, JI 161:2731-2739, 1998). The art has not addressednon-atopic responses.

IgE binds to two types of Fc receptors, called FcεRI (or high-affinityFcεR) (K_(a)=10¹¹ M⁻¹) and FcεRII (or low-affinity FcεR, CD23)(K_(a)<10⁸ M⁻¹). Therefore, unlike antibodies of the IgG class, IgEbinds to its FcR with extremely high affinity which in the case of FcεRIis about three orders of magnitude higher than that of IgG for the FcRs(FcγRI-III) and in the case of FcεRII is as high as that of IgG for itshigh affinity FcγRI (Gould, H J, et al., Annu. Rev. Immunol., 21:579-628. Epub@2001 Dec@19.:579-628 (2003); Gounni, A S, et al., Nature,367: 183-186 (1994); Kinet, J P, Annu. Rev. Immunol., 17: 931-72:931-972(1999) and Ravetch J V, and Kinet J P, Annu. Rev. Immunol., 9: 457-492(1991)). Because the IgE concentration in normal serum is usually verylow (less than 1 μg/mL), the FcεR are typically available for occupancyif IgE is induced by allergies and parasitic infestation or ifadministered. The FcεRI is composed of four polypeptide chains, one α,one β, and two γ chains. The α chain contains the IgE binding site andis a member of the immunoglobulin supergene family. The FcεRII consistsof one polypeptide chain which shows homology to animal lectinreceptors. FcεRI is expressed on mast cells and basophils as well asLangerhans cells and dendritic cells where it is involved in antigenpresentation, on eosinophils where it plays a role in defense againstparasitic infection, and also on monocytes (see Kinet, J P, Annu. Rev.Immunol., 17: 931-72.:931-972 (1999) for a review). Crosslinking of theFcεRI induces immediate release of mediators of inflammation such ashistamine, leukotrienes, prostaglandin E2, or β-glucuronidase anddelayed secretion of IL-4, 5, and 6. FcεRII is a member of the Igsuperfamily, more widely expressed on resting and mature B cells,monocytes, follicular dendritic cells, macrophages, eosinophils,platelets, Langerhans cells, and a subset of T cells (10-15% oftonsillar T cells). IL-4 up-regulates FcεRII expression on B cells andmacrophages. FcεRII on macrophages, eosinophils, and platelets mediatesADCC to schistosomules, enhance phagocytosis, and induce the release ofgranule enzymes (Gounni, A S, et al., Nature, 367: 183-186 (1994);Kinet, J P, Annu. Rev. Immunol., 17: 931-72.:931-972 (1999) andSpiegelberg, H L., J. Invest. Dermatol., 94: 49S-52S (1990)). FcεRII isinvolved in both IgE regulation and allergen presentation by B-cells,but understanding the functional roles of CD23 is further complicated bythe fact that it exists both as a cell surface molecule and in a solubleform generated by cleavage from the cell surface; furthermore, it existsin both monomeric and oligomeric states (see Gould, H J, and Sutton, BJ, Nat. Rev. Immunol., 8: 205-217 (2008) for a review). CD23 responds tohigh levels of IgE by downregulating IgE secretion. In human monocytes,CD23 triggering results in release of pro-inflammatory cytokinesincluding tumor necrosis factor (TNF)-α, IL-1, IL-6, andgranulocyte/macrophage-colony stimulating factor (GM-CSF). IL-4 appearsto play a central role in immediate-type hypersensitivity. It induceshuman B cells to secrete IgE and IgG4 and activated T helper cells. IL-4also stimulates mast cell growth and up-regulates FcεRII expression.

Most of the antibodies used in the treatment of cancer, including FDAapproved antibodies such as trastuzumab (HERCEPTIN®) and rituximab(RITUXAN®), are of the IgG class (Carter, P., IBC's Tenth InternationalConference. 6-9 Dec. 1999, La Jolla, Calif., USA. IDrugs, 3: 259-261(2000); Carter, P., Nat. Rev. Cancer, 1: 118-129 (2001) and Carter, P J,Nat. Rev. Immunol., 6: 343-357 (2006)). However, monoclonal IgEantibodies specific for tumor antigens have been reported. Theapplication of IgE for the therapy of cancer was pioneered by Nagy etal. (Nagy, E., et al., Cancer Immunol. Immunother., 34: 63-69 (1991)),who developed a murine IgE monoclonal antibody specific for the majorenvelope glycoprotein (gp36) of mouse mammary tumor virus (MMTV) anddemonstrated significant anti-tumor activity in C3H/HeJ mice bearing asyngeneic MMTV-secreting mammary adenocarcinoma (H2712) (Nagy, E., etal., Cancer Immunol. Immunother., 34: 63-69 (1991). Kershaw et al.(Kershaw, M H, et al., Oncol. Res., 10: 133-142 (1998)) developed amurine monoclonal IgE named 30.6, specific for an antigenic determinantexpressed on the surface of colorectal adenocarcinoma cells. Mouse IgE30.6 inhibited the growth of established human colorectal carcinoma COLO205 cells growing subcutaneously in severe combined immune deficient(SCID) mice, although this effect was transient. By contrast, a mouseIgG 30.6 and a mouse/human chimeric IgE 30.6 did not show anti-tumoreffects. The mouse IgE specific effect was attributed to the interactionof the antibody with FcεR bearing effector cells since the activity wasspecifically abrogated by prior administration of a nonspecific mouseIgE (Kershaw, M H, et al., Oncol. Res., 10: 133-142 (1998)). The lack ofeffect exhibited by the mouse/human chimeric IgE 30.6 is explained bythe fact that mouse FcεRI binds mouse IgE, but not human IgE. Gould etal. (Gould, H J, et al., Eur. J. Immunol., 29: 3527-3537 (1999))developed a mouse/human chimeric IgE (MOv18-IgE) and IgG MOv18 (IgG1)specific for the ovarian cancer tumor associated antigen folate bindingprotein (FBP). The protective activities of MOv18-IgE and MOv18-IgG1were compared in a SCID mouse xenograft model of human ovarian carcinoma(IGROV1). Mice were reconstituted with human peripheral bloodmononuclear cells (PBMC) to provide the model with effector cellscapable of binding human IgE constant regions. The beneficial effects ofMOv18-IgE were greater and of longer duration than those of MOv18-IgG1demonstrating the superior anti-tumor effects of IgE antibodies (Gould,H J, et al., Eur. J. Immunol., 29: 3527-3537 (1999)). In addition, thegroup of Gould et al. recently demonstrated for the first timemonocyte-mediated IgE-dependent tumor cell killing by two distinctpathways, ADCC and phagocytosis (ADCP), mediated through FcεRI andFcεRII (Karagiannis, S N, et al., Cancer Immunol. Immunother., 57:247-263 (2008) and Karagiannis, S N, et al., J. Immunol., 179: 2832-2843(2007)). This group has also used this assay system to assesspreliminary bioactivity of an anti-Her2 IgE construct, (Karragiannis P.,Cancer Immunol. and Immunother. 2008 epub ahead of print). Since humanPBMC are short-lived in SCID mice it is expected that the anti-tumoreffect will be enhanced in humans where the supply of effector cellswould be permanent. None of the studies could address the capacity ofthe mouse/human chimeric IgE to elicit an adaptive immune response dueto the fact that murine APCs such as Dendritic cells do not express theFcεRI (Kinet, J P, Annu. Rev. Immunol., 17: 931-72.:931-972 (1999)).

Relevant epidemiological studies on the association of allergic diseaseswith cancer support a lower risk of cancer among people with a historyof allergies or high levels of serum IgE including differenthematopoietic malignancies (Grulich, A E and Vajdic, C M, Pathology, 37:409-419 (2005); Wang, H. and Diepgen, T L, Allergy, 60: 1098-1111(2005); Grulich, A E, et al., Cancer Epidemiol. Biomarkers Prev., 16:405-408 (2007); Turner, M C, et al., Am. J. Epidemiol., 162: 212-221(2005); Wang, H. and Diepgen, T L, Br., J. Dermatol., 154: 205-210(2006); Wang, H., et al., Int. J. Cancer, 119: 695-701 (2006); Turner, MC, et al., Int. J. Cancer, 118: 3124-3132 (2006) and Melbye, M., et al.,J. Natl. Cancer Inst., 99: 158-166 (2007) and solid tumors such asovarian, colorectal, pancreatic cancer, and glioma (Wang, H. andDiepgen, T L, Allergy, 60: 1098-1111 (2005); Turner, M C, et al., Am. J.Epidemiol., 162: 212-221 (2005); Wang, H., et al., Int. J. Cancer, 119:695-701 (2006); Turner, M C, et al., Int. J. Cancer, 118: 3124-3132(2006); Mills, P K, et al., Am. J. Epidemiol., 136: 287-295 (1992);Wiemels, J L, et al., Cancer Res., 64: 8468-8473 (2004) and Wrensch, M.,et al., Cancer Res., 66: 4531-4541 (2006)).

Furthermore, mice infested with nematodes are resistant to syngeneicmammary adenocarcinoma and show lower incidence of spontaneous mammarytumors (Ogilvie, B M, et al., Lancet., 1: 678-680 (1971) and Weatherly,N F, J. Parasitol., 56: 748-752 (1970)). Eosinophilia, either inperipheral blood or tumor-associated tissue, is frequently associatedwith some tumor types and also found after immunotherapy with IL-2,IL-4, GM-CSF, and antibody to CTLA-4 (Lotfi, R, et al., J. Immunother.,30: 16-28 (2007). Within several tumor types including gastrointestinaltumors, this observation is associated with a significantly betterprognosis, whereas their presence in rejecting allografts is largelyseen as a harbinger of poor outcome (Lotfi, R. and Lotze, Mont., J.Leukoc. Biol., 83: 456-460 (2008)). Matta et al. (Clin Cancer Res.,13:5348-5354 2007) have reported that multiple myeloma patients withrelatively higher IgE levels had a better survival than patients withlower levels of IgE. Importantly, this is clearly reflected on thelevels of IgE and not the other classes of immunoglobulins. Thesestudies are consistent with a natural role of IgE in theimmunosurveillance of cancer including multiple myeloma. Fu, et al.(Clin Exp Immunol, 153:401-409, 2008) demonstrated that antibodies ofthe IgE class isolated from pancreatic cancer patients mediateantibody-dependent cell-mediated cytotoxicity (ADCC) against cancercells. Finally, treatment with omalizumab (XOLAIR®), which decreasesfree IgE in serum and down-regulates IgE receptors in effector cells todampen IgE-mediated inflammatory response, appears to lead to a higherchance of developing cancer. Approximately 1 in 200 treated asthmaticpatients developed breast, prostate, melanoma, non-melanoma skin, orparotid gland malignancies during the median observation period of 1year while in the control group the incidence was 1 in 500 (Dodig, S.,et al., Acta Pharm., 55: 123-138 (2005)). These studies suggest anatural role of IgE in the immunosurveillance of cancer with anestimated 186,320 new cases in the U.S. for 2008, prostate cancer is themost frequently diagnosed cancer (25% of all cancers) in men. Prostatecancer is the second leading cause of cancer deaths in American men,accounting for 10% (28,660 cases) of all cancer-related deaths. Forreasons that remain unclear, incidence rates are significantly higher inAfrican American men than in white men and death rates remain more thantwice as high as those in white men (Jemal, A., et al., CA Cancer J.Clin., 58: 71-96 (2008); Cancer Facts & Figures. American Cancer Society(2008) and Cancer Facts and Figures for African Americans 2007-2008.American Cancer Society, 2008). According to the most recent data, thelifetime probability of developing prostate cancer is 1 in 6. Over thepast 25 years, the 5-year survival rate for all stages combined hasincreased from 69% to almost 99% and relative 10-year and 15-yearsurvival is 91% and 76%, respectively. The dramatic improvements insurvival, particularly at 5 years, are mainly attributable to earlierdiagnosis (Cancer Facts & Figures, American Cancer Society (2008)). As aresult of the high survival rates, many patients die “with” theirdisease rather than “from” their disease, albeit following years ofinvasive therapies.

The high frequency of intercurrent mortality as opposed to prostatecancer-specific mortality, and the morbidity of currently availabletreatments for asymptomatic individuals, has led some groups to proposea less interventional or deferred-therapy approach for early-stagedisease (Cancer Facts & Figures, American Cancer Society (2008)). Morecontroversial is the management of tumors with adverse features such asa positive margin, vascular invasion, or capsular penetration. Mosturologists and radiation oncologists, however, do not recommendadditional therapy if the PSA is undetectable. If PSA persists, anddepending on the findings at surgery, the probability that the patienthas subclinical micrometastatic disease is high (DeVita, V T, et al.,Cancer—principles and practice of oncology (1997)). Clearly there is aneed for an effective, yet relatively benign treatment for men withpossible minimum disease (i.e., an effective cancer vaccine orimmunotherapy). For patients with metastatic disease or persistence ofPSA, which account for approximately 15% of patients (Cancer Facts &Figures, American Cancer Society (2008)), androgen ablation throughsurgical or pharmaceutical approaches often controls malignancy forextended periods; however, adverse effects are significant, andultimately, prostate cancer becomes hormone refractory. Chemotherapy andpalliative radiotherapies can help with patient management, but cannotcure this condition.

Prostate cancer is associated with two well characterized and highlyspecific antigens, prostate-specific antigen (PSA) and prostate-specificmembrane antigen (PSMA). PSA is a particularly attractive target antigenfor immunotherapy because this 33-kDa protein (serine protease) isalmost exclusively synthesized within the prostate gland and foundcirculating in human serum. PSA can also be identified in thecytoplasmic compartment of prostate epithelium and prostate tumor cellsand is released into the tumor microenvironment. The increasing serumlevels of PSA associated with the development of prostate cancer make itboth a useful marker for disease progression as well as a promisingtarget for immunotherapy, particularly T cell mediated immunotherapy(Zhang, S., et al., Clin. Cancer Res., 4: 295-302 (1998)). High levelsof PSA are found in the tumor (Katzenwadel, A. et al., Anticancer Res.,20: 1551-1555 (2000); Sinha, A A, et al., Anticancer Res., 19: 893-902(1999); Sinha, A A, et al., Anat. Rec., 245: 652-661 (1996) andMirochnik, Y., et al., Drug Deliv., 11: 161-167 (2004)). Vaccination inboth humans (Roos, A K, et al., Prostate, 62: 217-223 (2005) and mice(Pavlenko, M., et al., Br. J. Cancer, 91: 688-694 (2004)) with DNAencoding PSA has demonstrated specific immunity as measured byPSA-specific cytotoxic T lymphocytes (CTLs). In addition, this approachwas protective when mice were challenged with PSA-expressing tumors.Additionally, in a phase II clinical trial investigating theimmunogenicity of autologous DC pulsed with human recombinant PSA,specific immunity was generated in several patients as demonstrated bythe presence of PSA-specific T cells detected by enzyme-linkedimmunoSPOT (ELISPOT) (Barrou, B., et al., Cancer Immunol. Immunother.,53: 453-460 (2004)).

Given the profound medical impact of prostate cancer (28,660 expecteddeaths from prostate cancer for 2008), the relatively high mortalityrate for African American men, and the lack of adequate therapies formen with refractory metastatic disease, there is a need to developinnovative modalities of treatment in this indication.

SUMMARY OF THE INVENTION

The present invention provides therapeutic monoclonal IgE antibodiescomprising Fcε constant regions and variable regions comprising at leastone antigen binding region specific for binding a single epitope of acirculating, tumor-associated antigen (TAA) that is not a cell surfaceantigen wherein the epitope of the targeted antigen is not highlyrepetitive or is a non-repetitive epitope. The IgE antibodies of theinvention are capable of mediating an ADCC immune response, a Th1/Tc1type immune response, or both, when administered to a subject capable ofmounting such an immune response. Also provided are methods of inducingIgE-mediated immune responses against circulating TAA antigens that arenot cell surface antigens for use in inhibiting or killing tumorssecreting the target antigens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: IgE ELISA of purified antibody. Microwell Maxi-Sorp plates arecoated with PSA (1.5 ug/ml in PBS) and incubated at 4° C. overnight.After washing plates are blocked with 3% BSA/PBS for 30-60 minutes atRT. Samples containing anti-PSA IgE are added in serial dilutions (2ug/ml, 1 ug/ml, 0.5 ug/ml, 0.25 ug/ml 0.125 ug/ml 0.0625 ug/ml, 0.31ug/ml) and incubated for 2 hr at RT. The presence of anti-PSA specificIgE Abs is detected by biotinylated goat anti-Human IgE (1:2000dilution, KPL) followed by Strepavidin-HRP (1:10,000) added separatelyand incubated for 30-60 min each. The assay is developed with TMB andthe reaction is terminated with stop solution (H₂SO₄). Absorbance ismeasured at 405 nm.

FIG. 2: Demonstration of Local Accumulation of PSA in a tumor cell 3DCollagen cluster. A bar graph plots the signal of intensity to detectPSA using a goat anti PSA ELISA (coated overnight at 5 ug/mL). Spheroidmicrocultures of CT-26-d PSA transfectoma or CT-26-NEO control tumorcells lines are grown as hanging drops of 20 ul and pipetted into aPurecoll (Advanced Biomatrix) 3 mg/ml collagen extracellular matrixpreparation and grown for 72 hours. After washing, samples were added (5hanging drops containing 5×10⁴ cells cultured for four days, or 4 wellsof collagen, media, and microspheres that had been seeded with hangingdrops of 2.5 or 5×10⁴ cells cultured for 5 days). Samples were incubatedfor 2 hr at RT. Plates were again washed and AR47.47 (mouse anti-PSA, 5μg/ml) was added to each well and incubated for 1 hr at RT. AR47.47 wasdetected using an anti-mouse HRP conjugated Ab that was added to eachwell at a 1:8000 dilution and incubated at RT for 1 hr. After washing,developing solution (TMB) was added and the reaction was terminated withstop solution (H₂SO₄). Absorbance was measured at 450 nm on aspectrophotometer.

FIGS. 3A-F are read outs from flow cytometric analysis depicting thebinding of purified fluorescently labeled anti-PSA IgE to FcεRI a on thesurface of CHO-3D10 cells using flow cytometric analysis (FIG. 3 c) andcomparing to other anti-antigen IgE monoclonal antibodies (FIGS. 3D and3E) as well as human IgG1 antibody as a negative control (FIG. 3F).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel IgE antibodies to directhypersensitivity reactions and alter antigen processing to cancer whileavoiding systemic hypersensitivity reactions (e.g. systemicanaphylaxis). The present invention provides novel IgE monoclonalantibodies capable of inducing an IgE-mediated immune response in asubject against a circulating tumor-associated antigen that is not acell surface antigen. Such IgE monoclonal antibodies are able to directhypersensitivity reactions and alter antigen processing to cancer (i.e.tumor associated antigens) while avoiding systemic hypersensitivityreactions that may be harmful to the subject such as systemicanaphylaxis. Without being limited to any particular theory ofoperability, the inventors have discovered that IgE antibodies inaccordance with the invention will target a tumor that is secretingcirculating TAAs by two parallel pathways. The first pathway is bymediating bystander antibody-dependent cell-mediated cytotoxicity (ADCC)and also possibly by phagocytosis (ADCP) effects in the tumormicroenvironment due to the high local antigen density in the tumorenvironment. Local concentration gradients in tumor occur from naturalaccumulation adjacent to tumor cells, aggregates associated with matrixproteins that bind secreted PSA, and also in the case of necrotic orapoptotic tumor, from membrane bound PSA exposed to the extracellularspace from the fraction of disrupted cells. All these conditions willfacilitate FcεRI mediated local degranulation. It is believed thatsecretion by the tumor of the TAA into the extracellular space of thetumor microenvironment in the presence of the IgE antibodies of theinvention facilitates the cross linking of Fcε receptors (FcεR) tomediate mast cell and basophil degranulation and ADCC by eosinophils,macrophages, monocytes and other effectors thus resulting in tumor siteinflammation and local, but not systemic hypersensitivity reactions.With regard to cancer antigens (e.g. circulating TAA) that are not cellsurface antigens, it was previously believed that tumor cell killingthrough an ADCC or CDC mechanism was not expected for antigens notexpressed at the cell surface.

The second pathway is by a Th1/Tc1-type immune response or enhancedimmune response due to the uptake of immune complexes composed ofanti-antigen IgE and circulating tumor-associated antigen by antigenpresenting cells. It is believed that due to the expression of FcεR ondendritic cells the immune complexes comprising the tumor associatedantigen and the IgE will be processed to give rise to activated T and Bcells. Currently the state of the art teaches that when immune complexesconsisting of antigen and polyclonal IgE antibodies or IgE antibodies toa multi-epitopic allergen bind to antigen presenting cells in atopicindividuals as well as to mast cells or basophils, the FcεR would becrosslinked, which leads to cellular activation, local cytokineproduction favoring a Th2 biased immune response, activation of T helper2 (Th2) cells, and secretion of interleukin (IL)-4 and IL-5. Thus theart expects that the cytokines will subsequently induce Th2 immunity andlead to allergic inflammation including recruitment and activation ofeosinophils and other allergic inflammatory cells. Maurer, D., et al.,J. Immunol., 161: 2731-2739 (1998) and Maurer D., et al., J. Immunol.,154: 6285-6290 (1995). The art does not address immunity in acircumstance where the antigen is self tumor antigen and not anallergen.

However, the inventors have found that when immune complexes consistingof antigen and monoclonal IgE antibodies to a tumor antigen epitope thatis not highly repetitive or that is a non-repetitive epitope are boundby dendritic cells derived from a non atopic patient in an in vitro cellculture system, the dendritic cells express primarily Fc epsilon RII andproduce a T cell stimulation to the antigen that has characteristics ofa Th1/Tc1 immune response, with prominent induction of IFN-gammaproducing CD4 and CD8 antigen specific lymphocytes. Of particularimportance is the generation through this mechanism of specific andprotective CD8 IFN-gamma positive cytotoxic T-lymphocytes (CTLs)response against the antigen which will cause lysing of tumor cells inthe tumor microenvironment or at metastatic sites that express the tumorantigen in context of MHC class I.

Together, these mechanisms can result in an acute inflammation of thetumor microenvironment with subsequent tumor destruction. The presenceof dying tumor cells would further allow the effective uptake andpresentation of other tumor associated antigens by antigen presentingcells (APCs), such as dendritic cells resulting in an efficient primingof the adaptive immune response. While potentially very powerful indestroying antigen associated tumors, administration of IgE antibodiesraises the concerns of possible direct systemic immediatehypersensitivity. The approach of the present invention is distinguishedin that the IgE antibodies of the invention are monoclonal with singleepitope specificity to an epitope that is not highly repetitive, or is anon-repetitive epitope of the tumor-associated antigen. Systemicreactions such as systemic anaphylaxis are generally associated with apolyclonal IgE response, and production of T cell derived mast cellactivating factors that permit a local reaction to become systemic.Systemic reactions are associated with the vigorous crosslinkingassociated with a multi-epitopic polyclonal IgE response. Immunecomplexes comprising a circulating tumor-associated antigen (TAA) incombination with an antibody of the invention to a non-repetitiveepitope of the TAA will be relatively less likely to crosslink FcεR tothe level of systemic symptoms when bound to mast cells or granulocytesin circulation than compared to antigen specific polyclonal IgE.Furthermore, it is believed that the therapeutic dosage will be muchlower than that associated with IgG classes of antibody therapy againstcancer (e.g. trastuzumab (HERCEPTIN®) and rituximab (RITUXAN°)) due tothe high affinity of IgE to the FcεR.

A “therapeutic IgE antibody” of the invention (also referred to hereinas a “monoclonal IgE antibody of the invention”) is a monoclonalantibody that comprises Fc epsilon (ε) constant regions and alsocomprises variable regions comprising at least one antigen bindingregion specific for a circulating, tumor-associated antigen (TAA) thatis not a cell surface antigen The term “monoclonal IgE antibody of theinvention” further encompasses IgE monoclonal antibody derivativeswherein cytokines, chemokines or other immunomodulator proteins arefused to the IgE antibodies of the invention. The term “cancer antigen”as used herein can be any type of cancer antigen known in the art. Apreferred cancer antigen of the invention is a circulatingtumor-associated antigen (TAA) that is not a cell surface antigen.

Circulating tumor-associated antigens that are not cell surface antigensinclude any soluble antigen that is detectable in body fluid (e.g. bloodserum ascites, saliva or the like), that is of tumor origin and that hasbeen shed, secreted or otherwise released from the tumor, but that isnot expressed on the surface of the tumor as an intact protein directlydetectable by an IgE monoclonal antibody of the invention. The most wellknown of such antigens is prostate specific antigen (PSA), however otherorgan specific antigens produced by organ specific malignancies areposited to exist, but are not associated with popular populationscreening assays. Such alternative tumor antigens are also subject tothis invention.

The terms “monoclonal antibody” or “monoclonal antibodies” as usedherein refer to a preparation of antibodies of single molecularcomposition. A monoclonal antibody composition displays a single bindingspecificity and affinity for a particular epitope. The monoclonalantibodies of the present invention are preferably chimeric, humanized,or fully human in order to bind human Fc epsilon receptors when thesubject host is a human. Humanized and fully human antibodies are alsouseful in reducing immunogenicity toward the murine components of, forexample, a chimeric antibody, when the host subject is human.

The term “chimeric monoclonal antibody” refers to antibodies displayinga single binding specificity which have one or more regions derived fromone antibody and one or more regions derived from another antibody. Inone embodiment of the invention, the constant regions are derived fromhuman Fc epsilon (ε) (heavy chain) and human kappa or lambda (lightchain) constant regions. The variable regions of a chimeric IgEmonoclonal antibody of the invention are typically of non-human originsuch as from rodents, for example, mouse (murine), rabbit, rat orhamster.

As used herein, “humanized” monoclonal antibodies comprise constantregions are derived from human Fcε (heavy chain) and human kappa orlambda (light chain) constant regions. The variable regions of theantibodies preferably comprise a framework of human origin and antigenbinding regions of non-human origin.

Fully human or human-like antibodies may be produced through vaccinationof genetically engineered animals such as mouse lines produced atAbgenix Inc. (Thousand Oaks, Calif.) and MedaRex (Princeton, N.J.) whichcontain the human immunoglobulin genetic repertoire and produce fullyhuman antibodies in response to vaccination. Further, the use of phagedisplay libraries incorporating the coding regions of human variableregions which can be identified and selected in an antigen screeningassay to produce a human immunoglobulin variable region binding to atarget antigen.

The term “antigen binding region” refers to that portion of an antibodyof the invention which contains the amino acid residues that interactwith an antigen and confer on the antibody its specificity and affinityfor the antigen. The antibody region includes the “framework” amino acidresidues necessary to maintain the proper confirmation of the antigenbinding residues.

An “antigen” is a molecule or portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce antibody capable of binding to an epitope of that antigen. Anantigen can have one or more epitopes that are the same or different. Ina preferred embodiment, the antibodies of the invention are specific fora single epitope that is not highly repetitive, or a non-repetitiveepitope of the antigen. When the epitope is a non-repetitive epitope ofthe antigen, immune complex formed by an antibody of the invention andthe antigen is referred to as “monovalent” in that each antigen moleculemay be bound by only one antibody of the invention at any one time. Inone embodiment, the antigen is a capable of being bound by an IgEantibody of the invention to form an immune complex that is capable ofinducing a specific IgE-mediated immune response to the antigen in asubject capable of mounting such immune response. In one embodiment, theantigen, on its own, may not be capable of stimulating an immuneresponse for any number of reasons, for example, the antigen is a “self”antigen, not normally recognized by the immune system as requiringresponse or the immune system has otherwise become tolerant to theantigen and does not mount an immune response.

The term “epitope” is meant to refer to that portion of an antigencapable of being recognized by and bound by an antibody at one or moreof the antibody's binding regions. Epitopes generally comprisechemically active surface groupings of molecules such as amino acids orsugar side chains and have specific three dimensional structurecharacteristics as well as specific charge characteristics. An epitopethat is not “highly repetitive” means an epitope whose frequency andconfiguration upon the antigen are such that when an immune complex isformed between the IgE antibody and the antigen, such immune complexdoes not cause crosslinking of the Fcε receptors on dendritic cells orother relevant antigen presenting cells. The term “non-repetitiveepitope” means that only one such epitope is present in the antigen andthat the immune complex that is formed between the IgE antibody of theinvention and the antigen forms a monovalent immune complex.

An “immune complex” is a complex formed by an antibody and its targetantigen. An immune complex may be multimeric or monovalent as describedearlier. Methods for raising antibodies, such as murine antibodies to anantigen and determining if a selected antibody binds to a unique antigenepitope are well known in the art.

Screening for the desired antibody can be accomplished by techniquesknown in the art, e.g., radioimmunoassay, ELISA (enzyme-linkedimmunosorbant assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitin reactions, immunodiffusion assays, in situimmunoassays (using colloidal gold, enzyme or radioisotope labels, forexample), western blots, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention. For preparation of monoclonal antibodies, anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture may be used (see, e.g., Antibodies—ALaboratory Manual, Harlow and Lane, eds., Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, New York, 1988). These include but are notlimited to the hybridoma technique originally developed by Kohler andMilstein (1975, Nature 256:495-497), as well as the trioma technique,the human B-cell hybridoma technique (Kozbor et al., 1983, ImmunologyToday, 4:72), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additionalembodiment of the invention, monoclonal antibodies can be produced ingerm-free animals utilizing recent technology (PCT/US90/02545).According to the invention, human antibodies may be used and can beobtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad.Sci. U.S.A., 80:2026-2030) or by transforming human B cells with EBVvirus in vitro (Cole et al., 1985, in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, pp. 77-96). In fact, according to the invention,techniques developed for the production of “chimeric antibodies”(Morrison et al., 1984, J. Bacteriol. 159: 870; Neuberger et al., 1984,Nature 312:604-608; Takeda et al., 1985, Nature 314: 452-454) bysplicing the genes from a mouse antibody molecule specific for apolypeptide together with genes from a human antibody molecule ofappropriate biological activity can be used; such antibodies are withinthe scope of this invention.

In a preferred embodiment, the antibody of the invention is specific fora single, non-repetitive epitope of PSA. In one embodiment, the antibodyof the invention is specific for the epitope of PSA comprising aminoacids 139 to 163 EEFLTPKKLQCVDLHVISNDVCAQV (SEQ ID NO: 1) of PSA. Theexact epitope spans from amino acids 137-143 EPEEFLT (SEQ ID NO: 2) ofPSA. In one embodiment, the antibody of the invention is capable ofbinding PSA at the epitope defined at SEQ ID NO: 2. In one embodiment,the antibody of the invention is capable of binding all or any portionof the PSA epitope that extends from amino acids 135 to 163 having theamino acid sequence: SIEPEEFLTPKKLQCVDLHVISNDVCAQV (SEQ ID NO: 3). Theamino acid numbering used herein is based on the mature circulating PSAprotein that does not include the cleaved 24 amino acid leader sequence.

In a preferred embodiment, the antibody of the invention comprises thevariable regions of the light and heavy chain of MAb-AR47.47 and thehuman kappa or lambda (light chain) and human Fcε (heavy chain) constantregions. MAb-AR47.47 is a monoclonal murine IgG antibody that isspecific for the epitope expanding from amino acids 139 to 163 of PSA.Mab-AR47.47 is disclosed in detail in U.S. Pat. No. 6,881,405,incorporated herein by reference. Mab-AR47.47 is produced by a hybridomathat has ATCC Designation Number 1-18-12526. In one embodiment, the IgEmonoclonal antibody of the invention is specific for the epitope of PSAto which the monoclonal antibody MAb-AR47.47 also specifically binds.

In one embodiment, antibodies in accordance with the present inventionare constructed by genetically fusing the cDNA encoding the variableregions of the light and heavy chain of MAb-AR47.47 to the DNA encodingthe human kappa (light chain) and human Fcε (heavy chain) constantregions, respectively. The positive transfectoma identified byenzyme-linked immunosorbent assay (ELISA) and Western Blot, will becloned for highest productivity and selected for antibody production. Asused herein a “transfectoma” includes recombinant eukaryotic host cellsexpressing the antibody, such as Chinese hamster ovary (CHO) cells andNS/0 myeloma cells. Such transfectoma methodology is well known in theart (Morrison, S. (1985) Science, 229:1202). Previously publishedmethodology used to generate mouse/human chimeric or humanizedantibodies has yielded the successful production of various humanchimeric antibodies or antibody fusion proteins (Helguera G, Penichet ML., Methods Mol. Med. (2005) 109:347-74).

In general, chimeric mouse-human monoclonal antibodies (i.e., chimericantibodies) can be produced by recombinant DNA techniques known in theart. For example, a gene encoding the Fc constant region of a murine (orother species) monoclonal antibody molecule is digested with restrictionenzymes to remove the region encoding the murine Fc, and the equivalentportion of a gene encoding a human Fc constant region is substituted.(see Robinson et al., International Patent Publication PCT/US86/02269;Akira, et al., European Patent Application 184,187; Taniguchi, M.,European Patent Application 171,496; Morrison et al., European PatentApplication 173,494; Neuberger et al., International Application WO86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent Application 125,023; Better et al. (1988 Science,240:1041-1043); Liu et al. (1987) PNAS, 84:3439-3443; Liu et al., 1987,J. Immunol., 139:3521-3526; Sun et al. (1987) PNAS, 84:214-218;Nishimura et al., 1987, Canc. Res., 47:999-1005; Wood et al. (1985)Nature, 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst.,80:1553-1559).

The chimeric antibody can be further humanized by replacing sequences ofthe Fv variable region which are not directly involved in antigenbinding with equivalent sequences from human Fv variable regions.General reviews of humanized chimeric antibodies are provided byMorrison, S. L., 1985, Science, 229:1202-1207 and by Oi et al., 1986,BioTechniques, 4:214. Those methods include isolating, manipulating, andexpressing the nucleic acid sequences that encode all or part ofimmunoglobulin Fv variable regions from at least one of a heavy or lightchain. Sources of such nucleic acid are well known to those skilled inthe art and, for example, may be obtained from 7E3, ananti-GPII_(b)III_(a) antibody producing hybridoma. The recombinant DNAencoding the chimeric antibody, or fragment thereof, can then be clonedinto an appropriate expression vector. Suitable humanized antibodies canalternatively be produced by CDR substitution U.S. Pat. No. 5,225,539;Jones et al. 1986 Nature, 321:552-525; Verhoeyan et al. 1988 Science,239:1534; and Beidler et al. 1988 J. Immunol., 141:4053-4060.

In one embodiment, the immunogenicity of an IgE monoclonal antibody ofthe invention is reduced as compared to, for example, the parentantibody from which it was derived, using various strategies. Forexample, a chimeric IgE monoclonal antibody of the invention comprisinghuman Fcε constant regions and murine variable regions may be renderedless immunogenic to the human subject by genetically engineeringhumanized antibodies which comprise constant regions that are derivedfrom human Fcε and variable regions that comprise a framework of humanorigin and antigen binding regions of non-human origin that maintain thesame antigen specificity as that of the parent chimeric antibody.Alternatively, fully human or human like antibodies comprising the sameantigen specificity as the parent chimeric IgE monoclonal antibodies mayalso be genetically engineered using known procedures.

Other processes for reducing the immunogenicity of IgE monoclonalantibodies of the invention include, but are not limited to, processessuch as De-Immunization™ (Biovation Ltd., Aberdeen, United Kingdom andMerck KgaA, Darmstadt, Germany). This technology is a process thatidentifies murine epitopes present on murine or chimeric monoclonalantibodies that might cause immunogenicity in humans such as “humananti-mouse antibody” (HAMA) or “human anti-chimeric antibody” (HACA).This process further genetically alters these epitopes to avoid or atleast reduce immunogenicity as compared to antibodies that have not beensubjected to this process.

Other methods of reducing immunogenicity of monoclonal antibodies (Lazaret al., Mol. Immunol., 44, 1986-1998 (2007)) identifies framework andantigen binding region peptides or conformational motifs that mayactivate T-helper cells resulting in HAMA or HACA responses. Using thismethod a novel quantitative paradigm is used to determine the“humanness” of murine variable regions and murine regions of low humanidentity can be substituted for regions of higher human identity therebyreducing immunogenicity of the antibody.

In one embodiment an IgE monoclonal antibody of the invention isgenetically fused or chemically conjugated to a protein such as acytokine, chemokine or other immunomodulator protein. Methods forpreparing recombinant antibodies fused to various proteins are describedin U.S. Pat. No. 5,650,150. A review of antibody-cytokine fusionproteins is found at Helguera et al, Clinical Immunology, 105:233-246(2002); Helguera and Penichet, Methods Mol. Med. 109:347-74 (2005); andOrtiz-Sánchez et al, Expert Opin Biol Ther. 8(5):609-632 (2008).

In one embodiment, the immunomodulator is a cytokine selected from thegroup consisting of BDNF, CNTF, EGF, Epo, FGF, Flt3L, G-CSF, GM-CSF,I-309/TCA-3, gamma-IP-10, IFN alpha, IFN beta, IFN gamma, IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, LIF, LT, MCP-1 through MCP-3, M-CSF,MIF, MIP-1alpha, MIP-1beta, MIP-2, NGF, NT-3, NT-4, OSM, PBP, PBSF,PGFD, PF-4, RANTES, SCF, TGF alpha, TGF beta, TNF alpha, Tpo and VEGF.Suitable cytokines that are chemokines, can be selected from the groupconsisting of C10, EMF-1, ENA-78, Eotaxin, GCP-2, HCC-1, 1-309, IL-8,IP-10, Lymphotactin, MCP-1, MCP-2, MCP-3, MGSA, MIG, MIP-1alpha,MIP-1beta, MIP-2, NAP-2, PF4, RANTES, SCM-1 and SDF-1. The cytokineportion of the aforementioned immunomodulator can be the entire cytokineprotein amino acid sequence, or a fragment of such fusion proteinsufficient to elicit a cytokine-specific biological response.

Preferred immunomodulators or cytokines used to create fusion IgEantibodies of the invention include granulocyte/macrophage-colonystimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-12(IL-12), interferon gamma (IFNγ) and interferon alpha (IFNα).

A suitable junction between a cytokine polypeptide chain and an IgEmonoclonal antibody of the invention includes a direct polypeptide bond,a junction having a polypeptide linker between the two chains, or otherchemical linkage between the chains including the use of biotinylationand the avidin-biotin complex. In one embodiment, the junction is eithera direct or polypeptide linker spaced polypeptide linkage. This directlinkage allows for the expression of the immunotherapeutic agent as asingle fusion protein, from a host cell transformed with a suitableexpression vector encoding for the fusion protein immunotherapeuticagent.

In one embodiment, the IgE monoclonal antibody of the invention may bechemically conjugated to an immunomodulator that is a cytotoxic agent. Asuitable cytotoxic agent is one which has a direct cytotoxic effect onthe tumor cell, e.g. immunotoxins, radioisotopes, cytotoxic drugs, andthe like. Like cytokine immunomodulators described above, cytotoxicpeptides can be joined to the IgE monoclonal antibodies to form a fusionprotein either directly, or spaced by a linker peptide or chain.Chemical linkage of chemical cytotoxins to the IgE monoclonal antibodiescan be made. Radioisotope bearing IgE antibodies chains can also beconstructed. IgE monoclonal antibodies of the invention may also bechemically conjugated to other immunomodulators selected fromnon-protein, protein mimetic or synthetic therapeutic (e.g. smallmolecules or oligonucleotides) immunomodulators including but notlimited to: toll-like receptor agonists, immunostimulatoryoligodeoxynucleotides, cytokine blockers, and specific cytokine receptorantagonists.

The invention provides methods for inducing an IgE-mediated immuneresponse against a circulating TAA that is not a cell surface antigen ina subject capable of mounting said immune response comprisingadministering to the subject an effective amount of an IgE monoclonalantibody that specifically binds a single epitope of a circulatingtumor-associated antigen that is not a cell surface antigen, wherein theepitope is not highly repetitive against the tumor such that anIgE-mediated immune response is elicited. As used herein, a “subjectcapable of mounting (the referenced) immune response” is a subject suchas a human patient or other animal subject with functional T-cells, mastcells, eosinophils and dendritic cells with receptor affinity for theadministered IgE antibody of the invention as distinguished fromnon-human animal models, for example, whose immune systems do notcontain Fc epsilon receptors capable of binding human IgE permittinggeneration of functional T-cells, mast cells, eosinophils and dendriticcells in response to the administered antibody.

As used herein the induction of an IgE-mediated immune response includesone or more of the following:

i) Hypersensitivity against the antigen/IgE immune complex particularlyin the tumor micro-environment as evidenced by degranulation of mastcells and basophils bound to such immune complex via IgE antibodyreceptors FcεRI and/or FcεRII and the release of histamine, for example;

ii) Direct targeting of tumor cells via ADCC immune responses, ADCPimmune responses or both ADCC and ADCP immune responses against theantigen/IgE immune complex particularly in the tumor micro-environmentas evidenced by the stimulation of eosinophils, mast cells, basophils,and other cells to release pro-inflammatory cytokines, proteases andvasoactive lipid mediators (e.g. leukotrienes, prostaglandin D2, andplatelet activating factor when bound to the antigen/IgE immune complexvia IgE antibody receptors FcεRI and FcεRII;

iii) a cellular response as evidenced in part by the production ofT-cells that are specific for the antigen, the antigen/IgE antibodyimmune complex, or a peptide of the antigen complexed with MHC;

iv) a Th1/Tc1 immune response in response to challenge with theantigen/IgE antibody immune complex as evidenced, for example, by theproduction of CD8 IFN gamma positive T cells in response to the tumorantigen and tumor;

v) a humoral response as evidenced by production of antibodies againstthe antigen or the antigen/IgE immune complex.

As used herein an “effective amount” of an IgE monoclonal antibody ofthe invention is that amount sufficient to recognize and bind theepitope of the circulating TAA that is not a cell surface antigen andinduce, elicit, or enhance the referenced immune response in accordancewith the invention.

The invention also provides a method for inducing an IgE-mediated immuneresponse against a tumor that is associated with a circulating antigenthat is not a cell surface antigen in a subject capable of mounting sucha response comprising administering to the subject an effective amountof an IgE monoclonal antibody that specifically binds a single epitopeof the circulating antigen wherein the epitope is not highly repetitiveor is non-repetitive, and wherein an IgE mediated immune responseagainst the tumor is elicited.

As used herein the induction of an IgE-mediated immune response againsta tumor that is associated with a circulating antigen that is not a cellsurface antigen, is evidenced, in part, by any one of the following:

i) the inhibition of tumor growth and/or the facilitation of tumordestruction in whole or in part resulting from acute inflammation of thetumor environment and subsequent tumor inhibition/destruction viaeffector cells bearing human Fc epsilon receptors able tobind—monoclonal IgE antibody and direct ADCC immune responses, ADCPimmune responses or both ADCC and ADCP immune responses reactions to theantigen in the micro-environment;

ii) T cell response evidenced by the production of T-cells against thetumor antigen and secondarily additional antigens derived from the tumorand expressed in the context of MHC on the tumor cell resulting in tumorinhibition or tumor lysing; or

iii) T cell response against the tumor evidenced by the production of Tcells against other antigens associated with tumor cells that have beenlysed as above in (ii).

The invention also provides methods of inducing direct IgE-mediated ADCCimmune responses, ADCP immune responses, or both ADCC and ADCP immuneresponses to a circulating TAA that is not a cell surface antigen in asubject capable of mounting such an immune response comprisingadministering to the subject an effective amount of an IgE monoclonalantibody that specifically binds a single epitope of the circulatingantigen wherein the epitope is not highly repetitive or isnon-repetitive, and wherein an IgE mediated ADCC immune response andpossibly or optionally an ADCP immune response against the antigen iselicited. In a preferred embodiment, the ADCC immune response andpossibly or optionally an ADCP immune response is elicited in themicroenvironment of a tumor that is secreting the circulating TAA. Alocal concentration gradient capable of triggering effector cells is theproduct of secretion, complexation of local antigen with extracellularproteins, and release of membrane associated PSA from apoptotic andnecrotic tumor cells present in the tumor cluster In another embodimentthe ADCC immune response and possibly or optionally an ADCP immuneresponse is capable of causing the lysing and killing of tumor cellswithin the tumor microenvironment via bystander effects.

The invention also provides methods for inducing or enhancing a Th1/Tc1type immune response (particularly CD8 CTL response) to a circulatingTAA that is not a cell surface antigen comprising administering to asubject capable of mounting such response, an IgE antibody of theinvention that specifically binds a single epitope of a circulatingtumor-associated antigen that is not a cell surface antigen, wherein theepitope is not highly repetitive or is non-repetitive. As discussedabove, it is known from the allergy literature that polyclonal IgEantibodies to allergen induce a Th2-type immune response. In contrast,is believed that monoclonal IgE antibodies of the invention willprimarily produce Th1/Tc1 (e.g. a CD4, CD8 CTL, IFN gamma associatedcellular response) in the context of IgE directed to self antigen andalso the humoral immune response mediated by B cells. The effect of aprimary CD8 CTL response would be to mediate anti-tumor effects withreduced tendency to induce clinically worrisome immediatehypersensitivity in a patient such as systemic anaphylaxis. Additionallyor alternatively, the methods of the invention may reduce or eliminatesystemic hypersensitivity by reducing or eliminating crosslinking of Fcεreceptors which is the cause of mast cell or basophil degranulation.

The invention also provides a method for the treatment of cancerassociated with the antigen to which the antibody of the invention isspecific, by administering a composition comprising an IgE monoclonalantibody of the invention that specifically binds a single epitope of acirculating, tumor-associated antigen that is not a cell surfaceantigen, wherein the epitope is not highly repetitive. In oneembodiment, the invention provides a method of treating prostate cancerin a subject by administering to the subject a therapeutic IgEmonoclonal antibody of the invention that specifically binds a singleepitope of PSA wherein the epitope is not highly repetitive.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of an antibodyof the invention and a pharmaceutically acceptable carrier. In onepreferred embodiment, the pharmaceutical composition comprises atherapeutic IgE monoclonal antibody of the invention that specificallybinds a single epitope of PSA wherein the epitope is not highlyrepetitive in combination with a pharmaceutically acceptable carrier.

In one embodiment, the term “pharmaceutically acceptable” means approvedby a regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, and more particularly in humans. The term “carrier” refersto a diluent, adjuvant, excipient, or vehicle with which the therapeuticis administered. Such pharmaceutical carriers can be sterile liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like.

Water is a preferred carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of theantibody or fragment thereof, preferably in purified form, together witha suitable amount of carrier so as to provide the form for properadministration to the patient. The formulation should suit the mode ofadministration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where the composition is to be administered by infusion, it canbe dispensed with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the composition is administered byinjection, an ampoule of sterile water for injection or saline can beprovided so that the ingredients may be mixed prior to administration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the composition of the invention which will be effectivein the treatment, inhibition and prevention of the cancer associatedwith the antigen to which the antibody of the invention is specific andcan be determined by standard clinical techniques. In addition, in vitroassays may optionally be employed to help identify optimal dosageranges. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test systems.

For the antibodies of the invention, the dosage administered to apatient is typically 0.001 μg/kg to 1 mg/kg of the patient's bodyweight. Preferably, the dosage administered to a patient is between 0.01μg/kg and 0.1 mg/kg of the patient's body weight, more preferably 0.02μg/kg to 20 μg/kg of the patient's body weight. Generally, the IgEmonoclonal antibodies of the invention have a much higher affinity forthe Fcε R (as compared to IgG antibodies, for example) and longerhalf-life within the human body than antibodies from other species.Thus, lower dosages of the antibodies of the invention and less frequentadministration is often possible.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e. combined with other agents. For example acombination therapy can include a composition of the present inventionwith at least one anti-tumor agent, efficacy enhancing agent, and/orsafety enhancing agent.

The pharmaceutical compositions of the present invention have in vitroand in vivo diagnostic and therapeutic utilities. For example, thesemolecules can be administered to cells in culture, e.g., in vitro or exvivo, or in a subject, e.g., in vivo, to treat cancer. As used herein,the term “subject” is intended to include human and non-human animals. Apreferred subject is a human patient with cancer. As used herein theterms “treat” “treating” and “treatment” of cancer includes inhibitingthe onset of cancer in a patient; eliminating or reducing tumor burdenin a patient; prolonging survival in a cancer patient; prolonging theremission period in a cancer patient following initial treatment withchemotherapy and/or surgery; and/or prolonging any period between cancerremission and cancer relapse in a patient.

As used herein, “administering” refers to any action that results inexposing or contacting a composition containing an antibody of theinvention with a pre-determined cell, cells, or tissue, typicallymammalian. As used herein, administering may be conducted in vivo, invitro, or ex vivo. For example, a composition may be administered byinjection or through an endoscope. Administering also includes thedirect application to cells of a composition according to the presentinvention. For example, during the course of surgery, tumor cells may beexposed. In accordance with an embodiment of the invention, theseexposed cells (or tumors) may be exposed directly to a composition ofthe present invention, e.g., by washing or irrigating the surgical siteand/or the cells.

In accordance with a method of the invention compositions comprising theIgE monoclonal antibody of the invention may be administered to thepatient by any immunologically suitable route. For example, the antibodymay be introduced into the patient by an intravenous, subcutaneous,intraperitoneal, intrathecal, intravesical, intradermal, intramuscular,or intralymphatic routes. The composition may be in solution, tablet,aerosol, or multi-phase formulation forms. Liposomes, long-circulatingliposomes, immunoliposomes, biodegradable microspheres, micelles, or thelike may also be used as a carrier, vehicle, or delivery system.Furthermore, using ex vivo procedures well known in the art, blood orserum from the patient may be removed from the patient; optionally, itmay be desirable to purify the antigen in the patient's blood; the bloodor serum may then be mixed with a composition that includes a bindingagent according to the invention; and the treated blood or serum isreturned to the patient. The invention should not be limited to anyparticular method of introducing the binding agent into the patient.

When used for therapy for the treatment of cancer, the antibodies of theinvention are administered to the patient in therapeutically effectiveamounts (i.e. amounts effective to eliminate or reduce the patient'stumor burden or otherwise ameliorate the symptoms of cancer in thepatient). The antibodies of the invention and the pharmaceuticalcompositions containing them will normally be administered parenterally,when possible, at the target cell site, or intravenously.

In another embodiment, the IgE antibodies of the invention can beco-administered with a therapeutic agent, e.g., a chemotherapeuticagent, an immunosuppressive agent (e.g. Rituximab), an anti-inflammatoryagent, or can be co-administered with other known therapies, such asphysical therapies, e.g., radiation therapy, hyperthermia,transplantation (e.g., bone marrow transplantation), surgery, sunlight,or phototherapy. Such therapeutic agents include, among others,anti-neoplastic agents such as doxorubicin (adriamycin), cisplatinbleomycin sulfate, carmustine, chlorambucil, and cyclophosphamidehydroxyurea which, by themselves, are only effective at levels which aretoxic or subtoxic to a patient. Cisplatin is intravenously administeredas a 100 mg/m² dose once every four weeks and adriamycin isintravenously administered as a 60-75 mg/m² dose once every 21 days.

Pharmaceutical compositions of the present invention can include one ormore further chemotherapeutic agents selected from the group consistingof nitrogen mustards (e.g., cyclophosphamide and ifosfamide), aziridines(e.g., thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g.,carmustine and streptozocin), platinum complexes (e.g., carboplatin andcisplatin), non-classical alkylating agents (e.g., dacarbazine andtemozolamide), folate analogs (e.g., methotrexate), purine analogs(e.g., fludarabine and mercaptopurine), adenosine analogs (e.g.,cladribine and pentostatin), pyrimidine analogs (e.g., fluorouracil(alone or in combination with leucovorin) and gemcitabine), substitutedureas (e.g., hydroxyurea), antitumor antibiotics (e.g., bleomycin anddoxorubicin), epipodophyllotoxins (e.g., etoposide and teniposide),microtubule agents (e.g., docetaxel and paclitaxel), camptothecinanalogs (e.g., irinotecan and topotecan), enzymes (e.g., asparaginase),cytokines (e.g., interleukin-2 and interferon-.alpha.), monoclonalantibodies (e.g., trastuzumab and bevacizumab), recombinant toxins andimmunotoxins (e.g., recombinant cholera toxin-B and TP-38), cancer genetherapies, physical therapies (e.g., hyperthermia, radiation therapy,and surgery) and cancer vaccines (e.g., vaccine against telomerase).

The present invention is further illustrated by the followingnon-limiting examples.

Example 1 Preliminary Study Involving The Construction of an IgG1 AndIgG3 Chimeric Antibody with Human Constant Regions

The methodology used to generate mouse/human chimeric or humanizedantibodies that has yielded the successful production of various humanchimeric antibodies or antibody fusion proteins is described (Helguera,G. and Penichet, M L, Methods Mol. Med., 109: 347-374 (2005)). Achimeric version of MAb-AR47.47 is described in U.S. Pat. No. 6,881,405.Briefly, the plasmid construction originated from the RNA of the murinehybridoma AR47.47, extracted using the RNeasy Mini Kit (Qiagen,Valencia, Calif.). cDNA was then prepared using the SuperScript IIIFirst-Strand synthesis System for RT-PCR (Invitrogen, Carlsbad, Calif.).Both the heavy and light chain variable regions were amplified by PCRand cloned into the pCR-Blunt II-TOPO® vector using the Zero Blunt®TOPO® PCR Cloning Kit (Invitrogen). After sequence confirmation, themurine heavy chain variable region was then subcloned into twoexpression vectors, containing either the human IgG1 or human IgG3constant regions. Both heavy chain expression vectors contain thesequences for the C_(H)1, hinge, C_(H)2, and C_(H)3 domains under thecontrol of a CMV promoter. The murine heavy variable region was clonedin frame with the human C_(H)1 domain. The murine variable light chainwas subcloned into a single expression vector containing the human kappaconstant region under the control of a CMV promoter.

Expression in murine myeloma cells: Anti-PSA light chain producers werefirst developed by electroporation of either Sp2/0Ag14 or P3X63Ag8.653murine myeloma cells with the light chain expression vector. The bestlight chain producer was identified by ELISA and transfected with boththe heavy and light chain expression vectors (IgG3 and kappa expressionvectors or IgG1 and kappa expression vectors). It is our experience thatproduction of the light chain is a major limiting factor for optimalproduction and secretion of recombinant antibodies. For this reason,cotransfection is performed in order to maximize the production ofantibody by the myeloma cells. The clones were screened by ELISA for theproduction of either IgG1 or IgG3 antibodies.

Testing: Secretion of human antibody was screened by ELISA for humankappa and human gamma chain. The best producers were selected based onthe ELISA signal and colony size. The smallest colonies with the highestsignal were chosen and expanded for further characterization. Antibodiessecreted from various clones were tested for proper assembly, secretion,and molecular weight of the recombinant antibodies using ³⁵S-labelingand immunoprecipitation. Correctly assembled clones with highproductivity were then tested for binding to PSA by ELISA.

All three expression vectors were successfully constructed andtransfected into murine myeloma cells. Both the anti-PSA IgG3 and IgG1antibodies were properly assembled and secreted as evidenced by³⁵S-labeling and immunoprecipitation. In each clone, the full antibodycomplex shows the expected molecular weight (approximately 170 kDa forIgG3). As an example of reducing conditions, the immunoprecipitationresults of five clones of anti-PSA IgG1. All five clones show thepresence of both the heavy chain (approximately 50 kDa) and the lightchain (approximately 25 kDa). Antigen binding studies were performedusing the supernatants of transfectomas. Both anti-PSA IgG3 and IgG1demonstrate the ability to bind a PSA peptide that is recognized by themurine AR47.47 murine IgG1 antibody. Direct supernatants as well assupernatants from a non-targeting, negative control mouse/human chimericantibody were tested. Dilutions of the supernatants were also tested todemonstrate dose dependent binding of the antibody to the antigen. Thesupernatant of anti-PSA IgG3 demonstrates greater signal than the IgG1,which is due to higher productivity of the IgG3 clones compared with theIgG1 counterparts. Further studies with purified antibodies need to beconducted to directly compare antibody-binding capabilities.

Example 2 Construction of the Human/Mouse Chimeric Anti-PSA IgE Antibody

A cell line that produces mouse/human chimeric light chain (kappa) withthe variable regions of the AR47.47 light chain has already beenobtained by transfecting the murine myeloma cell lines P3X63Ag8.653 andSp2/0-Ag14 by electroporation with the mammalian expression vectorduring construction of the mouse/human chimeric IgG1 or IgG3 AR47.47 asdiscussed in Example 1.

The DNA encoding the variable light (VL) and heavy (VH) chain domains ofthe AR47.47 antibody have been sequenced and cloned already for thegeneration of the chimeric AR47.47 IgG1 and IgG3. The variable heavychain region was subcloned from the chimeric anti-PSA IgG1 vector andcloned into a human IgE expression vector (Chan et al, MolecularImmunology 37 (2000) 241-252) so that the variable region is in framewith the CH1 domain. This heavy chain was then co-transfected into thePSA light chain producer described above.

IgE antibody secreting clones were tested for presence of heavy (epsilonchain) and light chain (kappa chain) by ELISA. The best producers willbe selected based on the ELISA signal and colony size. The smallestcolonies with the highest signal will be chosen and expanded for furthercharacterization. Antibodies secreted from various clones will be testedfor proper assembly, secretion, and molecular weight of the recombinantantibodies using ³⁵S-labeling and immunoprecipitation. Correctlyassembled clones with high productivity will then tested for binding toPSA by ELISA as described in Example 1. The best clones will be furthercloned three times and adapted to protein-free media (Hybridoma-SFM,Invitrogen; or HyQ® SFM4MAb™, Hyclone), and grown in roller bottles forproduction of antibody for Further preclinical study. The antibody willbe purified via diafiltration (50 kDa cut-off) and immunoaffinitychromatography on a monoclonal anti-hIgE-Fc MAb (HP6029, HP6061) coupledto HiTrap columns (GE Healthcare).

Example 3 In Vitro Biochemical and Biological Characterization of theHuman/Mouse Anti-PSA IgE

Binding to PSA antigen: The anti-PSA IgE was assessed for initial PSAbinding by ELISA. Microwell Maxi-Sorp plates are coated with PSA (1.5ug/ml in PBS) and incubated at 4° C. overnight. After washing plates areblocked with 3% BSA/PBS for 30-60 minutes at RT. Samples containinganti-PSA IgE are added in serial dilutions (2 ug/ml, 1 ug/ml, 0.5 ug/ml,0.25 ug/ml 0.125 ug/ml 0.0625 ug/ml, 0.31 ug/ml) and incubated for 2 hrat RT. The presence of anti-PSA specific IgE Abs is detected bybiotinylated goat anti-Human IgE (1:2000 dilution, KPL) followed byStrepavidin-HRP (1:10,000) added separately and incubated for 30-60 mineach. The assay is developed with TMB and the reaction is terminatedwith stop solution (H₂SO₄). Absorbance is measured at 405 nm (FIG. 1).

In future studies, a transfectoma secreting anti-DNS IgE and anti-DNSIgG1 (Chan, L A, et al., Mol. Immunol., 37: 241-252 (2000)) will be usedas negative controls for specificity. The ELISA is developed fordetection of murine and human IgGs and will be adapted for the chimericIgE by optimizing a human IgE-specific secondary antibody(Sigma-Aldrich). Binding to cells can also be demonstrated by usingintracellular staining by flow cytometry or by a cell-based ELISA withconfluent layers of LNCaP cells (ATCC), or PSA- and neo-transfected CT26or P818 cell lines, for example. The cells will be fixed andpermeabilized, then blocked and incubated with the anti-PSA IgE as wellas anti-PSA IgG and non-specific IgE controls. Bound antibody will betraced with anti-human IgE-HRP and TMB substrate. Specific binding canfurther be demonstrated by western blots of LNCaP or CT26.PSA lysates incomparison to cell lines not expressing PSA (NIH:OVCAR-3, CT26.neo) asdescribed in Example 1.

Binding to Fc epsilon receptors: Binding of anti-PSA IgE to the humanhigh-affinity IgE receptor FcεRI was confirmed using the CHO3D10, ahuman FcεRI alpha chain transfected CHO cell line that binds human Fcepsilon and that has been used by other researchers to study the bindingof human IgE (Chan, L A, et al., Mol. Immunol., 37: 241-252 (2000)). Ascontrols (in parallel) the binding of the murine IgG1 and the chimerichuman IgG1 and IgG3 were tested. FIG. 3A-F shows binding of purified PSAIgE to FcεR1α on the surface of CHO-3D10 cells using flow cytometricanalysis. CHO-3D10 cells express FcεR1, but not FcεRII (CD23) asdemonstrated by flow cytometry (FIG. 3A and FIG. 3B). FIG. 3C showsbinding of monoclonal anti-PSA IgE to CHO-3D10 cells as detected by afluorescently labeled anti-IgE Ab and flow cytometric analysis. Theanti-PSA IgE binding is similar to other human monoclonal anti-antigenIgE Abs (FIG. 3D and FIG. 3E). The binding is IgE specific sincemonoclonal human IgG1 binding can not be detected (FIG. 3F).

In future studies, further cell types to be investigated include thecell line HMC-1 that expresses FcεRII but not FcεRI, the lymphoblastoidcell line RPMI 8866 or IM9, which are known to express FcεRII at highdensity (Mayumi, M., et al., Clin. Exp. Immunol., 71: 202-206 (1988)),the FcεRII-negative human B-lymphoblastoid cell line Daudi (all fromATCC), and isolated human monocytes, immature and maturedmonocyte-derived Dendritic cells (Berlyn, K A, et al., Clin. Immunol.,101: 276-283 (2001)). CHO cells and anti-DNS IgE and IgG will be used asfurther negative controls. Binding will be analyzed by flow cytometryusing FITC-labeled anti-human IgE and anti-human IgG reagents.

Discrimination of involved receptors on human dendritic cells will beperformed by inhibition with fluorescently labeled, commerciallyavailable antibodies to CD23 (FcεRII, clone D3.6, Biolegend) and FcεRI.The humanized monoclonal IgG antibody omalizumab (XOLAIR®, Genentech),binding to the receptor-binding region of IgE Fc, can also be used toassess Fcε receptor binding. Its affinity for IgE is extremely high,1.5×10¹⁰ M⁻¹; and this affinity is just high enough to enable it tocompete and block soluble IgE binding to FcεRI at high concentrations.Therefore, the concentration-dependence of the inhibition of theanti-PSA IgE will be indicative of the receptors involved.

IgE-mediated antigen uptake, trafficking, and presentation in dendriticcells: The inventors have established the methodology to study theuptake, trafficking, and presentation of PSA complexed with murine andchimeric anti-PSA IgG in human monocyte-derived dendritic cells (Berlyn,K A, et al., Clin. Immunol., 101: 276-283 (2001)) as well as in aHER2/neu system (la Cruz, J S, et al., Mol. Immunol., 43: 667-676(2006)), and this same technology will be used to study anti-PSA IgEcomplexed with PSA. Analysis of protein uptake and trafficking indendritic cells will be conducted by incubating the cells with PSA,labeled with Alexafluor 488 (green fluorochrome) and complexed withanti-PSA IgE or anti-PSA IgG at different concentrations, and over timeat 37° C. in tissue culture. Cells will also be incubated in parallelwith antigen alone, or combined with anti-DNS IgE or IgG(isotype-matched negative controls). Cells will then be harvested andanalyzed for uptake of fluorescent PSA using flow cytometry. For antigentrafficking studies, the cells will be fixed after antigen uptake,permeabilized, and treated with biotinylated rat monoclonal anti-mouseCD71 (anti-murine transferrin receptor, an early endosome marker),biotinylated monoclonal rat anti-mouse CD107a (anti-murine LAMP-1, alysosome marker) or rabbit polyclonal anti-H2-DM (H2-DM the murineequivalent of human HLA-DM serves as a marker for MHC class II),followed by streptavidin-Alexafluor 568 (red fluorochrome). Antigenuptake and colocalization will then be analyzed by confocal microscopy(la Cruz, J S, et al., Mol. Immunol., 43: 667-676 (2006)). Antigensinternalized through endocytosis in APCs traffic through severalvesicular compartments such as lysosome and H2-DM that leads to MHCclass II antigen presentation. However, dendritic cells that havedeveloped a membrane transport pathway of antigen delivery to thecytosol can also access the cytosolic antigen-processing machinery forMHC class I presentation (Rafiq, K., et al., J. Clin. Invest., 110:71-79 (2002) and Rodriguez, A., et al., Nat. Cell Biol., 1: 362-368(1999)). If MHC class I or II restricted T cell lines are not availablethat are specific for human PSA, the ability of human dendritic cells topresent the antigen will be studied using an in vitro antigenpresentation assay. Briefly, human dendritic cells will be generatedfrom monocytes (negative magnetic cell isolation kit) with IL-4 andGM-CSF, and incubated with PSA, anti-PSA IgE or immune complexes of PSAand anti-PSA IgE on Day 6. Anti-PSA IgG alone and as immune complex aswell as mock-loaded dendritic cells will serve as controls. Dendriticcells will be matured with LPS or TNF-α/IFN-α for 24 h, washed, andincubated with isolated autologous T cells for 1 week. T cells will berestimulated with freshly prepared and loaded dendritic cells for 3weekly rounds. Two to 4 h after the final stimulation, cells will beincubated with Brefeldin-A, and harvested 18 h later for intracellularcytokine staining T cells will be incubated with anti-CD3-FITC andanti-CD8-PE/Cy5, washed, permeabilized and then split into groups forstaining with anti-IFN-γ-PE, and anti-IL-4-PE or anti-IL-5-PE. The cellswill be analyzed by flow cytometry and the percentage of CD8+/cytokine+as well as CD8−(assuming CD4+)/cytokine+ cells calculated. Predominanceof IFN-γ production will indicate induction of a Th1-type response,whereas production of IL-4 or IL-5 is indicative of Th2-type andallergic responses.

Example 4 Assessment of Direct Anti-Tumor Effector Mechanisms

A. ADCC assays: Since human monocytes, macrophages, and eosinophils havebeen demonstrated to be critical mediators of chimeric IgE anti-tumorADCC in vitro and in vivo (Karagiannis, S N, et al., Eur. J. Immunol.,33: 1030-1040 (2003), Jensen-Jarolim E, et al., Allergy, 63:1255-66(2008), tests of the ability of these cells to elicit a cytotoxic effectin LNCaP models will be conducted using a Calcein AM release assay. TheCalcein AM release assay is performed equivalently to a ⁵¹Cr releaseassay, but using fluorescently labeled tumor cells (Invitrogen).Eosinophils will be isolated to >95% purity by Percoll gradientcentrifugation (density 1.082 g/ml) (GE Healthcare) followed byimmunomagnetic separation with anti-CD16-coated immunomagnetic beads asdescribed (Kayaba, JI 2001), and used for assays immediately. Monocyteswill be isolated using a magnetic negative monocyte isolation kit(Stemcell Technologies). Macrophages will be generated from frozen humanPBMC according to standard methods (Gersuk, G, et al., J. of ImmunolMethods 299:99-106 (2005)). Briefly, isolated PBMC will be washed 3×with PBS/EDTA and then resuspended a 2×10⁶ cells/ml in media (IMDMsupplemented with 10% human AB serum, glutamine and pen/strep).Monocytes are isolated by adherence to Falcon Primaria™ coated 24 welltissue culture plates. Cells are allowed to attach to plate for 3 h at37° C. and non-adherent cells are removed by shaking the plate (3000 rpmfor 2 min) and washing cells 2× with PBS/EDTA. The remaining adherentcells are resuspended in 1.5 ml of complete media containing an addition10% autologous human serum and 40 ng/ml rhM-CSF (R&D systems). Cells aregrown for 7-10 days and media is changed on days 4 and 7. In order toassess a bystander effect to a target antigen that is not expressed onthe tumor cell surface, but that is secreted into the tumormicroenvironment, a 3D culture system may be required for the tumorcells. Multicellular tumor spheroids are prepared using AlgiMatrix™ 3Dculture system (Invitrogen) following manufacturer's instructions.Briefly, multicellular spheroids are generated by seeding 2.5×10⁴ cellsin 30 μl into the middle of a dry sponge followed by culturing for 5-10days. Viable spheroids can be isolated by dissolving the AlgiMatrix 3DCulture System and used in an ADCC assay with Alamar Blue™ to assesscell viability.B. Antigen-mediated mast cell degranulation: Mast cell granule releaseassays will be performed using the rat basophilic leukemia cell lineRBL-sx38, which expresses human FcεRIα subunit and endogenous rat FcεRIα, β, and γ subunits. Approximately 10⁶ RBL-sx38 cells will be incubatedin the presence of varying concentrations of anti-PSA IgE complexed withdifferent amounts of PSA. Anti-DNS IgE complexed with DNS-BSA will beused as a positive control. Antibodies without antigen as controls willalso be used. The incubations will be done in 1 h at 37° C. Granulerelease will then be assessed by measurement of the 13-hexosaminidasepresent in the RBL-sx38 cell lysate vs. supernatant fractions (Chan, LA, et al., Mol. Immunol., 37: 241-252 (2000) and Karagiannis, S N, etal., Eur. J. Immunol., 33: 1030-1040 (2003)) or release of histamine asdescribed. Briefly, supernatants from triplicate cell samples will beseparated from the corresponding pellets by centrifugation of thesamples 30 min after activation. The pellets will be disrupted by threerounds of freeze thawing followed by sonication. The histamine contentof the acylated samples will be quantitated with a commercial ELISA(ICN) according to the manufacturer's protocol and the percentage ofhistamine released relative to histamine in the pellet determined (Ochi,H., et al., Proc. Natl. Acad. Sci. U.S.A., 97: 10509-10513 (2000)).Alternatively, human mast cells purified and generated from human cordblood over 6 weeks of culture in the presence of recombinant stem cellfactor (SCF), IL-6, and IL-10 (c-kit+, CD13+, low-level FcεIα, uniformtoluidine blue metachromasia) as described by the group of Dr. J. Boyce(Ochi, H., et al., J. Exp. Med., 190: 267-280 (1999)), will be used.Human mast cells can also be derived from peripheral blood CD34⁺ cellsfrom healthy adult volunteers using magnetic beads (StemcellTechnologies) and culture in StemPro-34 medium (Invitrogen) containing20% charcoal-filtered FCS, SCF (100 ng/ml), GM-CSF (10 pg/ml), and 4%conditioned medium from an immortalized B cell line over 4 weeks (Jiang,Y., et al., J. Immunol., 177: 2755-2759 (2006)).

C. Local Accumulation of PSA Around PSA Tumor Cells:

A 3-D culture system was developed using PSA- and neo-transfected CT26tumor cells (J. Frelinger) by creating multicellular spheroids grown inselective media (RPMI-1640, supplemented with 10% FBS, Pen/Strep,glutamine, and G418) and transferring them into a collagen matrix.Briefly, the multicellular spheroids were generated using the hangingdroplet method with a drop size of 20 μA, which seeds 2.5−5×10⁴ cells ineach spheroid. Cell solutions were pipetted onto the inside of a 96-wellplate cover, placed over a medium-filled plate, and incubated (5% CO₂,37° C.) for 5 days. The extracellular matrix (ECM) consisting of PureCol(3 mg/ml, Advanced Biomatrix) and 10×RPM1 (9:1) was prepared andneutralized with 0.1 N NaOH to a pH of 7.2-7.6. The ECM solution (75 μl)is pipetted into a sterile 96-well plate and the multicellular spheroidswere pipetted into this collagen gel and put in the incubator for 4-6hours (hr). Once the gel solidified, 75 μl of media was pipetted on topof the collagen to allow diffusion of nutrients into the ECM.Microspheroids were then kept in a sterile incubator until ready for use(from 1-6 days) and media was changed every 2 days. To determine thelocal accumulation of PSA around the hanging drop cultures and thecollagen containing microspheroids, an anti-PSA ELISA was performed.Briefly, ELISA plates were coated with goat anti-PSA Ab (5 ug/ml)overnight at 4° C. The next day, the plates were washed and blocked with3% FBS in PBS for 1 hr at room temperature (RT). After washing, sampleswere added (5 hanging drops containing 5×10⁴ cells cultured for fourdays, or 4 wells of collagen, media, and microspheres that had beenseeded with hanging drops of 2.5 or 5×10⁴ cells cultured for 5 days).Samples were incubated for 2 hr at RT. Plates were again washed andAR47.47 (mouse anti-PSA, 5 μg/ml) was added to each well and incubatedfor 1 hr at RT. AR47.47 was detected using an anti-mouse HRP conjugatedAb that was added to each well at a 1:8000 dilution and incubated at RTfor 1 hr. After washing, developing solution (TMB) was added and thereaction was terminated with stop solution (H₂SO₄). Absorbance wasmeasured at 450 nm on a spectrophotometer.

PSA is clearly produced and detectible in association with the CT-26 PSAmicrospheroids but not in a CT-26 control cell line. The positivecontrol is human PSA coated at a concentration of 1.5 μg ml to thecontrol well (FIG. 2).

D. Passive cutaneous anaphylaxis: Passive cutaneous anaphylaxis isperformed in transgenic mice expressing the human FcεRIα. Transgenicmice are shaved and injected intradermally in different regions of thedorsal side with 50 μl of 6 mg/ml histamine base (HollisterStier,Spokane, Wash.), 1 μg anti-PSA IgE, CT26-PSA or CT26-Neo microspheroidalone, 2 μg of crosslinker anti-human kappa antibody alone, 1 μganti-PSA IgE plus CT26-PSA or CT26-Neo microspheroid, or 1 μg anti-PSAIgE crosslinked with 2 μg of an anti-human kappa antibody. After 15 min,1% Evans Blue in 250 μl saline was injected i.v. Mice are sacrificed 20min later, and local cutaneous anaphylaxis was assessed visually by theblue leakage in the area surrounding the injection.E. In vivo anti-tumor effects using anti-PSA IgE: To study the abilityof In Vitro primed dentritic cells obtained from our transgenic FcεR1receptor humanized mice to stimulate the anti-tumor response in vivo,the dentritic cells will be inoculated (s.c.) into the human PSAtransgenic mice. This study is possible since the genetic background ofboth transgenic models is the same (BALB/c). Dentritic cells vaccinatedmice will be challenged sc or iv with a lethal dose of human PSAtransgenic syngeneic tumors (ie CT26-PSA). The anti-tumor activity willfurther be studied in mice that are human IgE, human FcεR and human PSAtriple transgenic. These mice are tolerant to human PSA and human IgEand allow the growth of syngeneic human PSA expressing tumor cells whilebeing responsive to the human IgE. These mice would be injected s.c.with 0.5−1×10⁶ PSA expressing tumor cells (i.e. CT26-PSA) to inducetumor formation. Treatment with anti-PSA IgE prior to and subsequent totumor cell implantation with and with out coordinated co-administrationof selected cytotoxic agents such as cyclophosphamide would be used toevaluate the ability of the anti-PSA IgE to limit tumorformation/growth.

Discussion: The anti-PSA IgE antibody of the invention, takes advantageof the expression of PSA in over 90% of prostate cancers; however, thetargeted antigen is secreted and not a trans-membrane tumor cellsurface. Therefore, mediation of ADCC and ADCP is not expected towardisolated tumor cells. However, it believed that high concentrations ofPSA in the extracellular tumor matrix as is discussed above in Example4C is sufficient to trigger ADCC mediated by eosinophils, monocytes, ormacrophages or to degranulate mast cells and basophils locally. However,due to the high affinity of the FcεR and the high density of thereceptor on mast cells and eosinophils, we expect that these cellsextravasate into the tumor highly loaded with anti-PSA (slowdissociation rate) and will crosslink their receptors when contactingmultiple PSA molecules in close proximity, like in the tumor stroma.While the monoclonal IgE is expected to be effective, the role ofcross-linking by polyclonal anti-PSA IgG can also be investigated.Furthermore, if the anti-PSA IgE/PSA immune complexes are processed toactivate a Th2-type response to PSA, then such polyclonal anti-PSA IgGwould be generated in vivo. The anti-PSA antibody with human Fc epsilonconstant regions is not expected to mediate direct acute immediatehypersensitivity reactions (via systemic mast cell and basophildegranulation) for the very reason that the antigen is not expressed onthe surface of cells and the antibody is targeting a single epitope ofPSA; and therefore, only generating monovalentimmune complexes. Theinventors believe that this approach should permit safe monoclonal IgEinfusion. If PSA-anti-PSA IgE immune complex would trigger Th1-type andCTL responses to PSA, then those effector cells would be able to lysePSA-expressing tumor cells via a MHC class I and/or II restrictedpathway. Either way, this novel IgE approach is expected to target theneoplastic cells with sufficient capacity to orchestrate an anti-tumorimmune response. Antibodies against the remaining murine epitopes on thechimeric anti-PSA IgE may induce unwanted cross-linking of thePSA-anti-PSA IgE complexes. Such human anti-chimeric antibodies (HACA),cross-linking IgE on mast cells and granulocytes may induce early andlate-phase activation, and thus, may pose a risk for immediatehypersensitivity reactions. Thus, it may be necessary and is possible tofully humanize the anti-PSA IgE according to well known procedures ifnecessary.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. It will also be understood that noneof the embodiments described herein are mutually exclusive and may becombined in various ways without departing from the scope of theinvention encompassed by the appended claims.

1. An IgE monoclonal antibody that specifically binds a single epitopeof a circulating, tumor-associated antigen that is not a cell surfaceantigen, wherein the epitope is not highly repetitive.
 2. The antibodyof claim 1 wherein the monoclonal antibody is a chimeric monoclonalantibody or a humanized monoclonal antibody.
 3. The antibody of claim 1having a constant region that is of human origin.
 4. The antibody ofclaim 1 having variable regions that are of human origin, non-humanorigin or any combination thereof.
 5. The antibody of claim 1 whereinthe epitope is a non-repetitive epitope.
 6. The antibody of claim 1wherein said antibody is capable of eliciting an IgE-mediated immuneresponse in a mammal.
 7. The antibody of claim 1 wherein thecirculating, secreted tumor-associated antigen is prostate-specificantigen (PSA).
 8. The antibody of claim 7 which binds to the epitope ofPSA defined by amino acids EEFLTPKKLQCVDLHVISNDVCAQV (SEQ ID NO: 1) ofPSA.
 9. The antibody of claim 7 which binds to the epitope of PSAdefined by amino acids SIEPEEFLTPKKLQCVDLHVISNDVCAQV (SEQ ID NO: 3) ofPSA or any portion thereof.
 10. The antibody of claim 7 wherein theantibody is a chimeric monoclonal antibody, or a fully humanizedmonoclonal antibody.
 11. A method for inducing an IgE-mediated immuneresponse against a circulating secreted tumor-associated antigen that isnot a cell surface antigen in a subject capable of mounting said immuneresponse comprising administering to the subject the antibody of claim 1in an amount effective to induce an IgE-mediated immune response in themammal.
 12. The method of claim 11 wherein the IgE-mediated immuneresponse is induced in the mammal in the absence of systemichypersensitivity reactions in the subject.
 13. The method of claim 11wherein the IgE-mediated immune response induced in the subject iscapable of inhibiting the progression of a tumor secreting the antigen.14. The method of claim 11 wherein the circulating, secreted, tumorassociated antigen is PSA.
 15. The method of claim 14 wherein theIgE-mediated immune response induced in the subject against PSA iscapable of inhibiting tumor growth or causing tumor destruction of atumor secreting PSA.
 16. A method of inducing a direct ADCC immuneresponse, a direct ADCP immune response, or both, to a circulating TAAthat is not a cell surface antigen in a subject capable of mounting sucha response comprising administering to the subject an effective amountof an IgE monoclonal antibody that specifically binds a single epitopeof the circulating antigen wherein the epitope is not highly repetitiveor is non-repetitive, and wherein a direct IgE mediated ADCC or ADCPimmune response against the antigen is elicited.
 17. The method of claim16 wherein the direct ADCC or ADCP immune response is elicited in themicroenvironment of a tumor that is secreting the circulating TAA. 18.The method of claim 16 wherein the direct ADCC or ADCP immune responseis induced in the absence of systemic hypersensitivity reactions in thesubject.
 19. The antibody of claim 7 which is genetically fused orchemically conjugated to a protein immunomodulator.
 20. A method oftreating prostate cancer in a subject comprising administering to thesubject a therapeutically effective amount of the IgE monoclonalantibody of claim 7.